This application contains subject matter which is related to that in concurrently filed application Ser. No. 392,951 entitled "Method For Improving Contrast in Surface Immunological Tests With Large Size Proteins" filed Aug. 30, 1973, now abandoned and patent applications Ser. No. 266,278 entitled "Method and Apparatus for Detection and Purification of Proteins and Antibodies" filed June 26, 1972 now abandoned and Ser. No. 384,113 entitled "Improved Method and Apparatus for Detection and Purification of Proteins and Antibodies" filed July 30, 1973, now abandoned all having a common applicant and assignee.
Other publications which relate to the present invention primarily as background are "Optical Measurement of the Thickness of a Film Adsorbed from a Solution", authors Irving Langmuir et al, Journal of the American Chemical Society, Vo. 59 (July - December 1937) page 1406; "Immunologic and Enzymatic Reactions Carried Out at a Solid-Liquid Interface" by Alexandre Rothen, Physiological Chemistry and Physics, 5, (1973), pages 243-258, "Interactions Among Human Blood Proteins at Interfaces", Leo Vroman et al, Federation Proceedings, Vo. 30, No. 5 (Sept. - Oct. 1971) pages 1494-1502, and "The Antibody-Antigen Reaction: A Visual Observation", Ivar Giaever, The Journal of Immunology, Vol. 110, No. 4 (May 1973) pages 1424-1426.
Immunological reactions are highly specific interactions in which an antigen interacts with a second biological constituent specific to the antigen and generally known as the antibody to form an immunological complex. Immunological reactions taking place within a biological system such as an animal or a human being are vital in combating disease. In a biological system, the entry of a foreign protein, i.e., the antigen, causes the biological system to produce the specific antibody proteins to the antigen in a process not fully understood at this time. The antibody protein molecules have available chemical combining or binding sites which complement those on the antigen molecule so that the antigen and antibody combine or bond to form the immunological complex.
Most antigens are proteins or contain proteins as an essential part, whereas all antibodies are proteins. Proteins are large molecules of high molecular weight, specifically, they are polymers consisting of chains of amino acids. The antigen and antibody protein may each have several combining sites. The five major classes of antibodies (immunoglobulins IgG, IgM, IgA, IgE, and IgD) are each apparently characterized by at least two heavy (long) peptide chains of amino acids and at least two light (short) peptide chains of the acids wherein the bond between the amino acid units is known as a peptide bond. These heavy and light peptide chains are oriented in the general shape of the letter "Y" and the active or combining sites are the extreme ends of the two arms of the Y-shaped antibody for the IgG antibody.
In addition to the immunological reaction which occurs between specific protein antigens and specific protein antibodies resulting in the formation of a protein antigen-protein antibody complex, other immunological complexing reactions between immunologically reactive antigens and antibodies are also contemplated by this invention. In addition, specific reactions between other biological particles, such as enzymes and substrates are also among the test methods contemplated and are embraced by the term "immunological reaction" as used herein. As used herein the terms "antigen" and "antibody" include enzymes and substrates and similar biological particles.
For instance, the following systems include biological particles which are capable of undergoing the immunological reactions described herein:
Viruses PA1 Bacteria and Bacterial toxins PA1 Fungi PA1 Parasites PA1 Animal tissue PA1 Animal body fluids, and the like. PA1 Trypsin extract - trypsin antibody PA1 chymotrypsin extract - chymotrypsin antibody PA1 pepsin extract - pepsin antibody PA1 ribonuclease extract - ribonuclease antibody PA1 thrombin extract - thrombin antibody PA1 amylase extract - amylase antibody PA1 penicillinase extract - penicillinase antibody PA1 insulin - insulin antibody
With respect to viruses, the antigens are viral cultures or parts thereof and the antibody specific thereto can be produced by administration of the antigens to a living host. Illustratively, antigen-antibody complexes in the following virus systems are useful in the herein disclosed procedure: Rubella virus culture (antigen) -- Rubella virus antibody; polio virus culture (antigen) -- polio virus antibody; vesicular stomatitis virus (VSV) culture (antigen) -- VSV antibody.
Regarding bacteria and bacterial toxins, the antigens are the particular bacteria or bacterial toxin or parts thereof and the antibody is produced by injection of the antigen into a living host. The following are illustrative examples of antigen-antibody pairs which can be used in the present method: tetanus toxoid suspension (antigen) -- tetanus antibody; diphtheria toxin suspension (antigen) -- diphtheria antibody; Neisseria gonorrhoeal suspension (antigen) -- gonorrhea antibody; Treponema pallidum suspension (antigen) -- syphilis antibody.
As for fungi, the antigens are antigenic extracts of fungal suspensions and the antibody is the fungal antibody produced by injection into a living host. Antigen-antibody complexes of fungi systems are illustrated by the following: Aspergillus extract suspension (antigen) -- aspergillus fungus antibody; Candida extract suspension (antigen) -- candida fungus antibody.
Antigens and antibodies in parasite systems are tested in a similar fashion to those of fungi. The system Toxoplasma gondii extract (antigen) -- Toxoplasma gondii antibody is a typical example.
By the term polysaccharides is meant a system wherein the antigen is a carbohydrate antigen. An example of such an antigen-antibody containing system is pneumococcus polysaccharides (antigen) -- pneumococcus antibody.
In addition to the typical enzyme - enzyme substrate reaction which is intended to be covered herein, enzymes themselves or parts thereof may be utilized an antigens and the antibody is the particular enzyme antibody elaborated by a living host after injection. Illustrative antigen-antibody complexes of enzyme systems are:
With respect to hormones, the antigenic constituent is usually found in a hormone extract and the antibody is the particular hormone antibody elaborated by the living organism after injection. An exemplary antigen-antibody complex is:
Although the ensuing discussion is directed for the most part to immunological interactions between specific protein antigens and specific protein antibodies, it is understood that it also applies to the systems and the immunologically reactive antigens and antibodies hereinabove described.
Immunological reactions can be detected by various techniques including the use of a suitable substrate such as a metallized glass or metal slide. Exposure of the substrate to an aqueous medium containing antigen will result in the antigen being physically adsorbed in a dense monomolecular layer onto the surface of the substrate. Subsequent exposure of the antigen-coated substrate to a serum containing antibodies specific to the antigen results in the immunological reaction wherein the antibodies selectively attach themselves to the antigens by means of the binding sites on the antibody molecule which complement those on the antigen molecule to thereby form at least a partial bimolecular layer of immunologically complexed antigen-antibody on the substrate surface. The problem arises in a subsequent exposure of the coated substrate containing the antigen-antibody complex to an aqueous medium containing, or suspected of containing, the same antigen. This subsequent exposure will generally result in no further binding of antigen to antibody since all of the active sites of the antibodies are already bound to the first antigenic layer due to the active antigenic molecules being very closely spaced together. Thus, there is a problem in surface immunology of the antibodies binding to a (antigen) surface and losing their activity (i.e., ability to further bind with an antigen, cell or virus).
Therefore, one of the principal objects of this invention is to provide a new method for binding antibodies to a surface so that the antibodies remain active.
A further object of this invention is to provide a new method for binding antibodies to an adsorbed antigen layer with combining sites of the antibodies remaining active for a further immunological reaction.
Another object of this invention is to provide a new method for forming multimolecular immunologically complexed films.